The present invention relates to a new procedure for the preparation of highly pure fractions of hyaluronic acid and its salts. Moreover, the invention encompasses a specific fraction of hyaluronic acid and its salts, in particular its sodium salt, obtainable by said procedure and having an average molecular weight within a specified range.
Hyaluronic acid (HA) is a typical and important representative of a class of biological macromolecules known as glycosaminoglycans (mucopolysaccharides). HA is a biological polymer which is present, with identical molecular structure, in all connective tissues of vertebrate organisms, where it plays a structural and biological role, in the sense that its local levels are strictly correlated with the tonus, trophism and tissue repair in case of injury. A review on the physiological role of these biological substances was given in Phys. Rev. (Comper W. D., Laurent T. C.: Physiological function of connective tissue polysaccharides. Phys. Rev., 58, (1), 255-315, 1978). The chemical-physical nature of HA is that of a saccharide biopolymer (D-glucuronic acid and N-acetylglycosamine), polymerized in alternation, forming long, unbranched molecular chains varying in molecular weight to a maximum of 8,000,000 Daltons (Meyer K.; Chemical Structure of Hyaluronic Acid. Fed. Proceed. 17, 1075, 1958; Laurent T. C.; Chemistry and Molecular Biology of Intracellular Matrix, 703-732, Academic Press N.Y., 1970). The behavior of this biopolymer in aqueous solution guarantees a particular viscosity, called visco-elasticity, which is typical of some biological fluids, such as synovial fluid and vitreous fluid, where HA is present at a concentration of 0.12-0.24% (Balazs E. A. et al.: Hyaluronic acid and replacement of vitreous and aqueous humor. Mod. Probl. Ophthal., 10, 3-21, 1972). Also aqueous humor, of human origin, was found to contain HA in an average concentration of 1.14 mcg/g (Laurent U. B. G.: Hyaluronate in human aqueous humor. Arch. Ophthalmol., 101, 129-130, 1983).
A body of published evidence has accumulated showing that the local supply of exogenous HA has distinct therapeutic and protective benefits in a great variety of pathological conditions of connective and epithelial tissues, such as:
impaired tissue regeneration in non-healing skin ulcers; PA1 arthrosic degeneration of articular connective tissue; PA1 ocular surgery. PA1 97% of the initial value (storage at 25.degree. C. for 6 months) PA1 75% of the initial value (sterilization at 118.degree. C. for 32 min.) PA1 80% of the initial value (sterilization at 121.degree. C. for 16 min.) PA1 90% of the initial value (sterilization at 124.degree. C. for 8 min.) PA1 0=normal PA1 +1=mild PA1 +2=moderate PA1 +3=severe or extensive PA1 0=clear PA1 1=slight haze--fundus visible PA1 2=moderate haze--fundus barely visible PA1 3=haze--red fundus reflex PA1 4=haze--gray fundus reflex PA1 5=fundus not visible PA1 a) presurgical evaluation, for example to assess PA1 b) Post-surgical evaluations, such as: PA1 The highest percentage of complications (in 7.9% of all patients) occurred between surgery and day 1, while smaller percentages were reported at days 7 and 30. PA1 At day 1 post-operatively the proportion of HEALON.RTM.-treated patients (14.3%) suffering complications was 2.5 times higher than for HA-1-treated patients (5.7%). This difference was statistically significant (p=0,079y by Fisher's two-tailed exact test). Differences on days 7 and 30 were not statistically significant (p&gt;0.6 by Fisher's two-tailed exact test). Complications included intra-ocular pressure rises to 30 mmHg or more ( 7/158=4.4% of HA-1-treated patients, 5/56=8.9% of HEALON.RTM.-treated patients) and corneal edema, iritis, conjunctivitis, hyphema, macular edema, wound leak, cyclitic membrane and subconjunctival hemorrhage. PA1 No significant differences in mean intra-ocular pressure occurred between treatment groups; however, HEALON.RTM.-treated patients had greater mean intra-ocular pressure rises at both 3 hrs and 1 day and had a significantly greater standard deviation of intraocular pressure than HA-1-treated patients at day 1. PA1 A slightly higher proportion of HEALON.RTM.-treated patients (37.0%) than HA-1-treated patients (28.6%) reported post-operative complications (Table 4). Complications included rises in intra-ocular pressure ( 5/42=11.9% of HA-1-treated patients, 11/46=23.9% of HEALON.RTM.-treated patients) and cases of hyphema, wound leak, vitreous hemorrhage, posterior capsule opacification, choroidal detachment, iris atrophy and macular edema. PA1 The two treatment groups did not differ statistically with respect to either mean intra-ocular pressures or variability of the intra-ocular pressure at baseline or at any time from day 1 to day 90 (Table 5). During the first 9 hours after surgery, it appears that HEALON.RTM. (removed from the eye) and HA-1 (left in place) produced post-operative intra-ocular pressure distribution which differed primarily with respect to standard deviations. HEALON.RTM.-treated patients had greater standard deviations and a significantly higher mean value at 1 hour post-operatively and HA-1-treated patients had greater, but only marginally significant standard deviations, at 6 and 9 hours post-operatively. At 1 hr post-operatively, 22.7% of HEALON.RTM.-treated patients and 10% of HA-1-treated patients had intraocular pressures of 21 mmHg or more. At 6 hours and 9 hours post-operatively, comparable proportions of HA-1-treated (73.2% at 6 hours and 65.0% at 9 hours) and HEALON.RTM.-treated (68.2% at 6 hours and 71.4% at 9 hours) patients had pressure readings of 21 mmHg or greater. PA1 No other difference was revealed between the two groups regarding the observations reported in the 1st study. Similarly, specular microscopy (endothelial cell count) revealed no significant differences (Tab. 6).
Particularly appreciated is the possibility, provided by the visco-elastic nature of HA, to coat the tissues exposed to risk of damage during surgical manipulation. According to surgeons who have used HA, the presence of a viscous layer of exogenous HA on the tissues which are most exposed to traumatizing accidental contacts, such as the cornea, exerts an efficient protective influence, which is reflected to a very positive degree in the successful outcome of the operation.
The protective effect and the facilitatory influence on tissue repair exerted by exogenous HA on the cornea has been shown both in experimental animals (Miller D. et al.: Use of Na-hyaluronate during intraocular lens implantation in rabbits. Ophthalmic Surgery, 8, (6), 58-61, 1977; Miller D. et al.: Use of Na-hyaluronate in autocorneal transplantation in rabbits. Ophthalmic Surgery, 11, (1), 19-21, 1980; Graue E. L. et al.: The protective effect of Na-hyaluronate to corneal endothelium. Exp. Eye Res., 31, 119-127, 1980; Ozaki L. et al.: Protective effect of Healon-coated intraocular lens on the corneal endothelium. Folia Ophthalmologica Japonica, 32, 1301-1305, 1981) and in man (Norm M.: Preoperative protection of cornea and conjunctiva. Acta Ophthalmologica, 59, 587-594, 1981; Polack F. M. et al.: Sodium hyaluronate (Healon) in keratoplasty and IOL implantation. Ophthalmology, 88, 425-431, 1981).
Several procedures are known for the preparation of purified hyaluronic acid and of particular fractions with a high degree of purity to be used in therapy, for example in the aforesaid indications.
The molecular fractions of integral hyaluronic acids obtained directly by extraction of organic materials, for example from hens' crests, have molecular weights which can vary within wide limits, for example from about 90% -80% to 0.2% of the molecular weight of the integral acid, preferably between 5% and 0.2%. The fractions can be obtained from the integral acids with hydrolyzing or oxidizing or enzymatic chemical agents or physical procedures, for example mechanical or by irradiation, and often, therefore, primordial extracts are formed in these same purification procedures (see for example the article by Balazs et al. in "Cosmetics & Toiletries". Italian edition No. 5/84, pp 8-17). The separation and purification of the molecular fractions obtained is effected by the known techniques.
For example, in U.S. Pat. No. 4,141,973, a procedure is described for the preparation of hyaluronic acids with a molecular weight of at least 750,000 Daltons which can be used, in view of their particular and high degree of purity and for the absence of inflammatory effects, in operations on the eye. The procedure consists of extracting the PEA sodium salt from the starting material, eliminating blood residues from the animal organs used, deproteinizing the extract thus obtained, eliminating inflammatory impurities, treating the product in aqueous solution with a sterilizing agent and in precipitating the hyaluronic acid salt from the aqueous solution with an organic solvent. The blood residues are eliminated with ethanol, HA in the form of its sodium salt (which is the form in which it is found in the starting materials) is extracted with water, deproteinization is effected by treatment with diluted acids and simultaneous extraction of the hydrolyzed parts with chloroform, or by means of proteolytic enzymes, harmful inflammatory substances are eliminated by extraction with chloroform at pH 6-7 and sterilization is effected by treatment with cetylpyridinium chloride. By this procedure, the only hyaluronic acid fraction obtained is that specifically described in the patent as having a molecular weight of 1,586,000 Daltons. On the basis of its chemical, physical and biological properties, this molecular fraction of hyaluronic acid seems to correspond to the commercial product known by the trademark HEALON.RTM..
New techniques have been developed, such as molecular ultrafiltration. By this means of purification it is possible to discard those HA fractions with a molecular weight coming within the higher or lower margins of the range of molecular sizes. For example, in the EPO patent No. 01238572, granted on 25.7.1990, a procedure is described to obtain fractions of sodium hyaluronate with mean molecular weights of between 250,000 and 350,000 Daltons, by exposing the product directly obtained by extraction of organic material and subsequent enzymatic deproteinization with papain, to two molecular ultrafiltrations through membranes with a molecular cutoff of 30,000, that is, with membranes which trap only those fractions with molecular weights of over 30,000. This fractioning appears to be important to the obtainment of a product free from secondary actions of an inflammatory nature, since the fractions responsible for such effects are those with low molecular weights, for example about 30,000 Daltons. After further molecular filtration, using membranes with an exclusion limit of 200,000 (that is, membranes which trap those fractions with molecular weights of over 200,000 Daltons) the obtained filtered product is a fraction (called in the patent HYALASTINE) with a mean molecular weight between 50,000 and 100,000 Daltons, while the portion left on the membrane is a sodium hyaluronate fraction which has a mean molecular weight between 500,000 and 730,000 (the fraction called HYALRCTIN).